A promising area of cancer research is the discovery of cancer specific tumor-associated antigens (TAA). Screening the sera of patients having particular cancers against proteomic libraries, to detect autoantibody binding, can lead to the discovery of panels of TAA which can be used for early diagnosis, guiding therapy and developing anticancer vaccines. However, proteomic screens are difficult to perform using conventional technology such as protein microarrays which are difficult to produce, expensive to use and have limited scalability to larger protein numbers. During Phase I, we will evaluate a novel low-cost, high throughput approach for discovery of TAA signatures based on using Libraries of in vitro Expressed Proteins Displayed on Beads (LIVE-PDB). These human protein libraries can be produced inexpensively in single solid-phase PCR and cell-free protein expression reactions. LIVE-PDB is then utilized to screen patient sera for tumor-associated autoantibodies, hence facilitating the discovery of TAA signatures for early detection of specific cancers, and even their probability of recurrence. A key feature of LIVE- PDB is the ability to detect and decode the interactions of a patient's circulating TAA-specific autoantibodies with the entire proteomic library by using a massively parallel DNA sequencing platform. Initial work at AmberGen which facilitates this novel approach includes: i) the demonstration of single-molecule solid-phase emulsion PCR (SMSP-ePCR) on beads to form a Library of in vitro Expressible Genes Displayed on Beads (LIVE-GDB), ii) subsequent cell-free protein expression of the beads, which enables the inexpensive production of a proteome-scale LIVE-PDB, iii) parallel bead decoding by sequencing iv) the use of dual-epitope tagged cell-free expressed antigens in a proprietary T2-ELISATM assay which provides improved immuno-detection for TAA analysis and ultimately for clinical TAA based diagnostics and v) demonstration that in vitro expressed proteins provide a well-characterized, uniform source of antigens that interact with autoantibodies in human sera. We propose to evaluate this new approach during Phase I by creating a prototype LIVE-PDB starting with a commercially available Open Reading Frame template DNA library. Successful single-molecule solid-phase emulsion PCR on beads from this library to form the LIVE-GDB will be evaluated by massively parallel sequencing using commercially available services from 454 Life Sciences (Branford, CT). We will also evaluate LIVE-PDB for the detection of TAA in colorectal cancer (CRC). The project will be done in collaboration with Paul Schroy, Director of Clinical Research for the Section of Gastroenterology at the Boston Medical Center. Serum samples from CRC patients will be profiled by LIVE-PDB. Sensitivity and specificity of the LIVE-PDB screens will be determined by comparison to established reference assays for individual TAA. In Phase II, the approach will be expanded to proteome-scale LIVE-PDB and will be fully integrated onto the 454 Life Sciences platform, which will be adapted for discovering proprietary diagnostic TAA signatures. PUBLIC HEALTH RELEVANCE: A promising area of cancer research is the discovery of cancer specific tumor-associated antigens (TAA) which can be used for detecting cancer earlier, optimizing therapy and developing anticancer vaccines. We will evaluate a novel low-cost, high throughput approach for discovery of TAA signatures based on the fabrication of a proteome bead library. An important feature of this bead library is the ability to perform TAA discovery using a new generation of commercially available DNA sequencers which will speed-up the discovery process.